Determination of the presence and quantity of creatine kinase in biological fluids, particularly in human blood serum, has become very useful in the diagnosis of myocardial infarctions.
Conventional procedures for creatine kinase determination generally involve the consideration that creatine kinase catalyzes both the forward and reverse reactions illustrated by the equation:
creatine+adenosine triphosphate.revreaction.creatine phosphate+adenosine diphosphate. Both the forward and reverse reactions have been used in analytical procedures, but use of the reverse reaction is preferred because it is about 6 times faster than the forward reaction.
In one known analytical procedure carried out in solution, creatine and adenosine triphosphate (hereinafter ATP) are incubated with the liquid sample to be assayed. After a suitable time, the conversion of creatine to creatine phosphate is stopped by adding an acid to the sample. The acid hydrolyzes only the creatine phosphate to provide free inorganic phosphate which is measured colorimetrically as a directly proportional measure of creatine kinase activity. This procedure, while providing a simple and direct measure of creatine kinase activity, requires both an undesirably large sample volume to obtain a measurable amount of phosphate and unacceptable incubation times (e.g. up to 1 hour). Because of the strong reverse reaction of the equation hereinabove, adenosine diphosphate (hereinafter ADP) begins to inhibit the forward reaction as its concentration increases over the long incubation time. Further, this procedure does not allow for continuous kinetic monitoring of creatine kinase.
Other known procedures for measuring creatine kinase in solution involve coupling two or more enzyme reactions together as described, for example, in N. W. Tietz (Ed.), Fundamentals of Clinical Chemistry, W. B. Saunders Co., 1970, pp. 466-470. One measurement technique described in that reference is illustrated on page 467 in equations 45a-45c. The desired measurable product of the reaction sequence is nicotinamide adenine dinucleotide phosphate (reduced form, hereinafter NADPH) the presence of which is measured at 340 nm with a spectrophotometer. This procedure, however, is extremely pH-sensitive and subject to considerable error if strict pH control is not maintained. Further, NADPH and NADP.sup.+ (oxidized form) are relatively unstable. It would be desirable to avoid UV assay procedures because they require relatively complicated instrumentation and are subject to interferences from various serum components when creatine kinase activity is measured.
It is also known that creatine kinase is unstable in biological fluids apparently due to sulfhydryl (i.e. mercapto) oxidation and disulfide formation. Hence, an activator is commonly employed in solution assays to restore full creatine kinase activity. Sulfhydryl compounds such as thioglucose, dithiothreitol, dithioerythritol, mercaptoethanol, N-acetylcysteine and glutathione are among the most common activators used in the analytical procedures known in the art, e.g. procedures wherein changes in UV absorbance are measured. However, although it would be highly desirable to use colorimetric assay procedures, it has been found that when activators are used in high enough concentrations for desired creatine kinase activation, they adversely affect many chromogens (also known as colorimetric indicators) useful in colorimetric assays. Generally, the activators bleach such chromogens so that the color density is reduced and the assay then becomes unreliable.
For example, in U.S. Pat. Nos. 4,241,178 (issued Dec. 23, 1980 to Esders et al); and 4,089,747 (issued May 16, 1978 to Bruschi) a colorimetric assay method for measuring glycerol and a composition for detecting hydrogen peroxide, respectively, are described. The detectable colorimetric changes brought about by the described method are obtained from the use of chromogens (e.g. dyes, dye precursors, dye formers etc.) which can provide color changes when reacted with a peroxide. But it has been found that the chromogens described therein are undesirably bleached when used with the mercapto-containing activators noted hereinabove at concentrations generally required in known solution assays.
Hence, there is a need for a colorimetric method for determining creatine kinase in aqueous fluids which method avoids the disadvantages of known methods. Such disadvantages include the need for large volume samples for carrying out assay procedures. A colorimetric method is also desired which utilizes a reaction sequence involving stable reagents and byproducts which provides detectable and reproducible colorimetric changes, thereby avoiding the problems presented by known UV assay
procedures. It is further desired that such a method be suitable for both solution and dry assay procedures.